Soluble phosphatidylserine triggers assembly in solution of a prothrombin-activating complex in the absence of a membrane surface

Factor X(a) (FX(a)) binding to factor V(a) (FV(a)) on platelet-derived membranes containing surface-exposed phosphatidylserine (PS) forms the "prothrombinase complex" that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FV(a), FV(a1) and FV(a2). These two isoforms differ by a 3-kDa polysaccharide chain (at Asn(2181) in human FV(a1) (Kim, S. W., Ortel, T. L., Quinn-Allen, M. A., Yoo, L., Worfolk, L., Zhai, X., Lentz, B. R., and Kane, W. H. (1999) Biochemistry 38, 11448-11454)) and have different coagulant activities. We examined the interaction of the two bovine isoforms with active site-labeled FX(a), finding no significant difference. A soluble form of PS (C6PS) bound to FV(a1) and FV(a2) with comparable affinities (K(d) = 11-12 microm) and changes in FV(a) intrinsic fluorescence. At concentrations well below its critical micelle concentration, C6PS binding to bovine FV(a2) enhanced its affinity for FX(a) in solution by nearly 3 orders of magnitude (K(d)(eff) = 40-2 nm over a C6PS range of 30-400 microm) but had no effect on the affinity of FV(a1) for FX(a) (K(d) = 1 microm). This results in a soluble complex between FX(a) and FV(a2), whose expected molecular weight was confirmed by calibrated native gel electrophoresis. This complex behaved as a normal Michaelis-Menten enzyme in its ability to produce thrombin from meizothrombin (apparent k(cat)/K(m) congruent with 10(9) m(-1) s(-1)). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.     

The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex

Factors V(a) and X(a) (FV(a) and FX(a), respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential "prothrombinase" complex of blood coagulation. The C-terminal domain (C2) of FV(a) (residues 2037-2196 in human FV(a)) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp(2063) and Trp(2064). Mutating these tryptophans abolishes FV(a) membrane binding. To address both the roles of these tryptophans in C6PS or membrane binding and the role of the C2 domain lipid binding site in regulation of FV(a) cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFV(a2)-C2) and of a B domain-deleted factor V light isoform (rFV(a2)) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFV(a2)-C2 binds to C6PS and to 20% PS/PC membranes with apparent K(d) values of 2.8 microM and 9 nM, respectively, while mutant rFV(a2)-C2 does not. Equilibrium dialysis confirmed that mutant rFV(a2)-C2 does not bind to C6PS. Mutant rFV(a2) binds to C6PS (K(d) approximately 37 microM) with an affinity comparable to that of wild-type rFV(a2) (K(d) approximately 20 microM), although it does not bind to PS/PC membranes to which wild-type rFV(a2) binds with native affinity (K(d) approximately 3 nM). Both wild-type and mutant rFV(a2) bind to active site-labeled FX(a) (DEGR-X(a)) in the presence of 400 microM C6PS with native affinity (K(d) approximately 3-4 nM) to produce a solution rFV(a2)-FX(a) complex of native activity. We conclude that (1) the C2 domain PS site provides all but approximately 1 kT of the free energy of FV(a) membrane binding, (2) tryptophans lining the C2 lipid binding pocket are critical to C6PS and membrane binding and insert into the bilayer interface during membrane binding, (3) occupancy of the C2 lipid binding pocket is not necessary for C6PS-induced formation of the FX(a)-FV(a) complex or its activity, but (4) another PS site on FV(a) does have a regulatory role.     

Prothrombin activation on membranes with anionic lipids containing phosphate, sulfate, and/or carboxyl groups

Factor Xa catalyzed prothrombin activation is strongly stimulated by the presence of negatively charged membranes plus calcium ions. Here we report experiments in which we determined the prothrombin-converting activity of phosphatidylcholine (PC) membranes that contain varying amounts of different anionic lipids, viz., phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylmethanol (MePA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidyl-beta-lactate (PLac), sulfatides (SF), sodium dodecyl sulfate (SDS), and oleic acid. All anionic lipids tested were able to accelerate factor Xa catalyzed prothrombin activation, in both the absence and presence of the protein cofactor Va. This shows that the prothrombin-converting activity of negatively charged membranes is not strictly dependent on the presence of a phosphate group but that lipids which contain a carboxyl or sulfate moiety are also able to promote the formation of a functionally active prothrombinase complex. In the absence of factor Va, the prothrombin-converting activity of membranes with MePA, PG, PE, PLac, SF, or SDS was strongly inhibited at high ionic strength, while the activity of PS- and PA-containing membranes was hardly affected by ionic strength variation. This suggests that in the case of the ionic strength sensitive lipids electrostatic forces play an important role in the formation of the membrane-bound prothrombinase complex. For PS and to a lesser extent for PA we propose that the formation of a coordinated complex (chelate complex) with Ca2+ as central ion and ligands provided by the gamma-carboxyglutamic acid residues of prothrombin and factor Xa and the polar head group of phospholipids is the major driving force in protein-membrane association. Our data indicate that the anionic lipids used in this study can be useful tools for further investigation of the molecular interactions that play a role in the assembly of a membrane-bound prothrombinase complex. Membranes that were solely composed of PC can also considerably enhance prothrombin activation in the presence of factor Va. This activity of PC is only observed on membranes which are composed of PC that contains unsaturated hydrocarbon side chains. Membranes prepared from phosphocholine-containing lipids with saturated hydrocarbon side chains such as dimyristoyl-PC, dipalmitoyl-PC, distearoyl-PC, and dioctadecylglycerophosphocholine hardly accelerated prothrombin activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative roles of factor V(a) and phosphatidylserine-containing membranes as cofactors in prothrombin activation

Activation of prothrombin, as catalyzed by the prothrombinase complex (factor X(a), enzyme; factor V(a) and phosphatidylserine (PS)-containing membranes, cofactors), involves production and subsequent proteolysis of two possible intermediates, meizothrombin (MzII(a)) and prethrombin 2 plus fragment 1.2 (Pre2 & F1.2). V(max), K(m), or V(max)/K(m) for all four proteolytic steps was determined as a function of membrane-phospholipid concentration. Proteolysis was monitored using a fluorescent thrombin inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants for the conversion of MzII(a) and Pre2 & F1.2 to thrombin were determined directly. Pre2 & F1.2 conversion was linear in substrate concentration up to 4 microm, whereas MzII(a) proteolysis was saturable. First order rate constants for formation of MzII(a) and Pre2 & F1.2 could not be determined directly and were determined from global fitting of the data to a parallel, sequential model, each step of which was treated by the Michaelis-Menten formalism. The rate of direct conversion to thrombin without release of intermediates from the membrane-V(a)-X(a) complex (i.e. "channeling") also was adjusted because both the membranes and factor V(a) have been shown to cause channeling. k(cat), K(m), or k(cat)/K(m) values were reported for one lipid concentration, for which all X(a) was likely incorporated into a X(a)-V(a) complex on a PS membrane. Comparing previous results, which were obtained either with factor V(a) (Boskovic, D. S., Bajzar, L. S., and Nesheim, M. E. (2001) J. Biol. Chem. 276, 28686-28693) or with membranes individually (Wu, J. R., Zhou, C., Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (2002) Biochemistry 41, 935-949), with results presented here we conclude that both factor V(a) and PS-containing membranes induce similar rate increases and pathway changes. Moreover, we have determined: 1) factor V(a) has the greatest effect in enhancing rates of individual proteolytic events; 2) PS-containing membranes have the greatest role in increasing the preference for the MzII(a) versus Pre2 pathway; and 3) PS membranes cause approximately 50% of the substrate to be activated via channeling at 50 microm membrane concentration, but factor V(a) extends the range of efficient channeling to much lower or higher membrane concentrations.     

Role of procoagulant lipids in human prothrombin activation. 2. Soluble phosphatidylserine upregulates and directs factor X(a) to appropriate peptide bonds in prothrombin.

Activation of human prothrombin to thrombin (II(a)) by factor X(a) during blood coagulation requires proteolysis of two bonds and thus involves two possible activation pathways (parallel-sequential activation model). Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). A soluble lipid, dicaproylphosphatidylserine (C6PS), enhances activation by 60-fold [Koppaka et al. (1996) Biochemistry 35, 7482]. We report here that C6PS binding to factor X(a) not only enhances the rate of activation but also alters the pathway. Activation was monitored using a chromogenic substrate (S-2238) to detect both II(a) and MzII(a) active site formation and SDS-PAGE to detect Pre2-F1.2 as well as II(a) and MzII(a). Of the four kinetic constants needed to describe activation, two (MzII(a) and Pre2-F1.2 consumption) were measured directly, and two (MzII(a) and Pre2-F1.2 formation) were obtained by fitting the three time courses simultaneously to the parallel-sequential reaction model. The time courses of II(a), MzII(a), and Pre2-F1.2 formations were all well described below the C6PS critical micelle concentration (CMC) by this activation model. The rate of Arg(322)-Ile cleavage leading to MzII(a) formation increased by 150-fold, while the rate of Arg(273)-Thr cleavage leading to Pre2-F1.2 formation was inhibited slightly. At concentrations of water-soluble C6PS above its CMC, all four proteolytic reactions increased in rate by 2-5-fold at the C6PS CMC. We conclude that soluble C6PS differentially affects the rate of individual bond cleavages during prothrombin activation in solution such that activation occurs almost exclusively via MzII(a) formation. Finally, C6PS enhanced the rates of all proteolytic reactions to within a factor of 3 of the enhancement seen with PS-containing membranes. We conclude that PS-containing membranes regulate prothrombin activation by factor X(a) mainly via interaction of individual PS molecules with factor X(a).     

Notecarin D binds human factor V and factor Va with high affinity in the absence of membranes

Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ～55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ～5,000- and ～80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ～85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding.     

Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles.

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of prothrombinase assembly and activity by phosphatidylethanolamine

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (K(d) = ～10 nm) in the absence of phosphatidylserine (PS). These membranes support formation of a 60-70% functional prothrombinase complex at saturating factor Va concentrations. Although reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va(2) (K(d) = ～6.5 μm) and to factor Xa (K(d) = ～91 μm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (K(d)(app) = ～40 nm) of a partially active prothrombinase complex between factor Xa and factor Va(2), compared with K(d)(app) for C6PS ～2 nm. These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.     

Role of the N-terminal epidermal growth factor-like domain of factor X/Xa

The functional importance of the N-terminal epidermal growth factor-like domain (EGF-N) of factor X/Xa (FX/Xa) was investigated by constructing an FX mutant in which the exon coding for EGF-N was deleted from FX cDNA. Following expression and purification to homogeneity, the mutant was characterized with respect to its ability to function as a zymogen for either the factor VIIa-tissue factor complex or the factor IXa-factor VIIIa complex and then to function as an enzyme in the prothrombinase complex to catalyze the conversion of prothrombin to thrombin. It was discovered that EGF-N is essential for the recognition and efficient activation of FX by both activators in the presence of the cofactors. On the other hand, the FXa mutant interacted with factor Va with a normal apparent dissociation constant and activated prothrombin with approximately 3-fold lower catalytic efficiency in the prothrombinase complex. Surprisingly, the mutant activated prothrombin with approximately 12-fold better catalytic efficiency than wild-type FXa in the absence of factor Va. The mutant was inactive in both prothrombin time and activated partial thromboplastin time assays; however, it exhibited a similar specific activity in a one-stage FXa clotting assay. These results suggest that EGF-N of FX is required for the cofactor-dependent zymogen activation by both physiological activators, but it plays no apparent role in FXa recognition of the cofactor in the prothrombinase complex.     

Efficacy of soluble phospholipids in the prothrombinase reaction

The prothrombinase complex is comprised of an enzyme, factor Xa, and a cofactor, factor Va, that each bind peripherally to membranes containing phosphatidylserine (PS) and activate the substrate, prothrombin. The mechanism by which the membrane contributes to enhanced catalytic efficacy of prothrombinase is not precisely known but is generally attributed to some aspect of enzyme and substrate assembly on the multisite surface of the membrane. A recent proposal has suggested a radically different role in which individual phospholipid molecules, either in the membrane or as single soluble molecules, act by an entirely allosteric mechanism that does not involve the multisite feature of the membrane [Zhai, X., Srivastava, A., Drummond, D. C., Daleke, D., and Lentz, B. R. (2002) Biochemistry 41, 5675-5684]. Our study measured prothrombinse activity in the presence of phospholipids such as short-chain phosphatidylserine and lysophosphatidylserine (lyso-PS). Both enhanced prothrombinase activity, and the increase was consistent with the requirement for extended bilayer structure. Even then, prothrombinase activity was low when compared with activity on bilayer membranes of mixed PS and phosphatidylcholine (PC). Lyso-PS approached the activity of PS/PC membranes only when it was mixed with PC bilayers. The results suggest that the two-dimensional membrane bilayer surface is necessary for the support of full prothrombinase activity.     

Exposure of platelet membrane phosphatidylserine regulates blood coagulation

This article addresses the role of platelet membrane phosphatidylserine (PS) in regulating the production of thrombin, the central regulatory molecule of blood coagulation. PS is normally located on the cytoplasmic face of the resting platelet membrane but appears on the plasma-oriented surface of discrete membrane vesicles that derive from activated platelets. Thrombin, the central molecule of coagulation, is produced from prothrombin by a complex ("prothrombinase") between factor Xa and its protein cofactor (factor V(a)) that forms on platelet-derived membranes. This complex enhances the rate of activation of prothrombin to thrombin by roughly 150,000 fold relative to factor X(a) in solution. It is widely accepted that the negatively charged surface of PS-containing platelet-derived membranes is at least partly responsible for this rate enhancement, although there is not universal agreement on mechanism by which this occurs. Our efforts have led to an alternative view, namely that PS molecules bind to discrete regulatory sites on both factors X(a) and V(a) and allosterically alter their proteolytic and cofactor activities. In this view, exposure of PS on the surface of activated platelet vesicles is a key regulatory event in blood coagulation, and PS serves as a second messenger in this regulatory process. This article reviews our knowledge of the prothrombinase reaction and summarizes recent evidence leading to this alternative viewpoint. This viewpoint suggests a key role for PS both in normal hemostasis and in thrombotic disease.     

Proexosite-1-dependent recognition and activation of prothrombin by taipan venom

An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate prothrombin to thrombin. To determine whether, similar to prothrombinase, taipan venom utilizes proexosite-1 on prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg(35), Lys(36), Arg(67), Lys(70), Arg(73), Arg(75), and Arg(77)) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the K(m) and k(cat) values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr(63)-sulfated hirudin(54-65) peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a K(D) of approximately 2 microm, it is ineffective in inhibiting the activation of mutant zymogens (K(D) > 4-30 microm). Interestingly, an approximately 50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom.     

The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction

The activation of bovine prothrombin by prothrombinase (Factor Xa, Factor Va, synthetic phospholipid vesicles, and calcium ion) was studied in the presence of the fluorescent, reversible thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA). Recordings of fluorescence intensity during prothrombin activation exhibited maxima that decreased to stable limiting values. These data suggested the transient appearance of the meizothrombin-DAPA complex, which exhibits fluorescence with 1.5-fold greater intensity than the thrombin-DAPA complex. At substrate concentrations well below Km, progress curves could be fitted by equations describing an ordered, sequential conversion of prothrombin to thrombin through the intermediate meizothrombin via two pseudo-first order steps. The pseudo-first order rate constants for both steps varied linearly with enzyme concentration, indicating that both steps are catalyzed by prothrombinase. The progress of the reaction was also monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry analyses of aliquots removed at intervals spanning the reaction. These analyses confirmed both the existence of meizothrombin and its time course as predicted from the equations used to analyze fluorescence intensity profiles. Meizothrombin levels peaked at about 0.3 mol/mol initial prothrombin under the conditions typically studied. In addition, prethrombin 2, which is the intermediate expected from cleavages occurring in the order opposite that required to form meizothrombin, was not observed under any of the conditions examined. These data indicate that prothrombin activation catalyzed by the fully assembled prothrombinase complex proceeds via an ordered, sequential reaction with meizothrombin as the sole intermediate.     

Characterization of a factor Xa binding site on factor Va near the Arg-506 activated protein C cleavage site

Prothrombin is proteolytically activated by the prothrombinase complex comprising the serine protease Factor (F) Xa complexed with its cofactor, FVa. Based on inhibition of the prothrombinase complex by synthetic peptides, FVa residues 493-506 were proposed as a FXa binding site. FVa is homologous to FVIIIa, the cofactor for the FIXa protease, in the FX-activating complex, and FVIIIa residues 555-561 (homologous to FVa residues 499-506) are recognized as a FIXa binding sequence. To test the hypothesis that FVa residues 499-505 contribute to FXa binding, we created the FVa loop swap mutant (designated 499-505(VIII) FV) with residues 499-505 replaced by residues 555-561 of FVIIIa, which differ at five of seven positions. Based on kinetic measurements and spectroscopic titrations, this FVa loop swap mutant had significantly reduced affinity for FXa. The fully formed prothrombinase complex containing this FVa mutant had fairly normal kinetic parameters (k(cat) and K(m)) for cleavage of prothrombin at Arg-320. However, small changes in both Arg-320 and Arg-271 cleavage rates result together in a moderate change in the pathway of prothrombin activation. Although residues 499-505 directly precede the Arg-506 cleavage site for activated protein C (APC), the 499-505(VIII) FVa mutant was inactivated entirely normally by APC. These results suggest that this A2 domain sequence of the FVa and FVIIIa cofactors evolved to have different specificity for binding FXa and FIXa while retaining compatibility as substrate for APC. In an updated three-dimensional model for the FVa structure, residues 499-505, along with Arg-506, Arg-306, and other previously suggested FXa binding sequences, delineate a continuous surface on the A2 domain that is strongly implicated as an extended FXa binding surface in the prothrombinase complex.     

Role of procoagulant lipids in human prothrombin activation. 1. Prothrombin activation by factor X(a) in the absence of factor V(a) and in the absence and presence of membranes.

Activation of prothrombin by factor X(a) requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). Activation by human factor X(a) of human prothrombin was examined in the absence of factor V(a) and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (25:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzII(a), and Pre2-F1.2; a synthetic substrate (S-2238) detected thrombin or MzII(a) active site formation; and SDS-PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS-PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzII(a) and Pre2-F1.2 were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzII(a) active site formation and Pre2 appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M(-1) s(-1), for the rate of MzII(a) formation. By contrast, the parallel-sequential model could not describe prothrombin activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting prothrombin directly to thrombin without release from the membrane-enzyme complex. The data suggest that PS membranes (1) regulate factor X(a), (2) alter the substrate specificity of factor X(a) to favor the meizothrombin intermediate, and (3) "channel" intermediate (MzII(a) or Pre2-F1.2) back to the active site of factor X(a) for rapid conversion to thrombin.     

Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30–40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.     

Contribution of regions distal to glycine-160 to the anticoagulant activity of tissue factor pathway inhibitor

The functions of the first two Kunitz domains of tissue factor pathway inhibitor (TFPI) are well defined as active site-directed inhibitors of factor VIIa and factor Xa. The anticoagulant properties of the third Kunitz domain and C-terminal region were probed using altered forms of TFPI. TFPI-160 contains the first two Kunitz domains. K1K2C contains the first two Kunitz domains and the basic C-terminus. Neither TFPI-160 nor K1K2C contains the third Kunitz domain. In amidolytic assays containing calcium, TFPI-160 is a less potent inhibitor of factor Xa than TFPI. However, addition of the C-terminus in K1K2C nearly restores inhibitory activity to that of TFPI, indicating that the third Kunitz domain is not required for direct inhibition of factor Xa. When compared in assays containing phospholipids and factor Va, K1K2C and TFPI-160 are poor inhibitors compared to TFPI, demonstrating that the third Kunitz domain is required for the full anticoagulant activity of TFPI. TFPI was further characterized in amidolytic assays performed with Gla-domainless factor Xa and in prothrombin activation assays using submicellar concentrations of short-chain phospholipids (C6PS). TFPI and K1K2C are worse inhibitors of Gla-domainless factor Xa, compared to wild-type factor Xa, while TFPI-160 inhibits both forms of factor Xa equally, suggesting a C-terminus/Gla domain interaction. TFPI is a potent inhibitor of thrombin generation by prothrombinase assembled with C6PS, while TFPI-160 and K1K2C are not. Conversely, TFPI does not inhibit prothrombin activation by prothrombinase assembled on a two-dimensional lipid bilayer. Together, the data indicate that the region between Gly-160 and the end of the third Kunitz domain contributes to TFPI function by orienting the second Kunitz domain so that it can bind the active site of phospholipid-associated factor Xa prior to prothrombinase assembly and/or by slowing formation of the prothrombinase complex.     

Fibroblasts, glial, and neuronal cells are involved in extravascular prothrombin activation.

A membrane-associated prothrombin activator (MAPA) was found on various cultured cells derived from non-hematopoietic cells [Sekiya, F. et al. (1994) J. Biol. Chem. 269, 32441-32445]. In this study, we investigated the enzymatic properties of this enzyme using protease inhibitors. While the metalloproteinase inhibitor, o-phenanthroline, had no effect, some Kunitz type serine protease inhibitors attenuated MAPA activity. Recombinant tissue factor pathway inhibitor (rTFPI) also markedly reduced the activity (IC(50), 1. 3+/-0.6 x 10(-10) M). MAPA activity is, therefore, most likely to be due to factor Xa. We evaluated the effect of exogenous factor Xa on MAPA activity. Factor Xa-dependent prothrombin activation was observed on fibroblast cells (apparent K(d), 1.47+/-0.72 nM). Activation was also observed on glial and neuronal cells, which expressed MAPA activity. These results imply that membrane-bound factor Xa results in MAPA activity on these cells. Therefore, we considered the involvement of factor Va, a component of prothrombinase, in this activity. We examined whether or not the prothrombinase complex is assembled on these cells. Prothrombin was activated in a manner dependent on both exogenous factor Xa and factor Va (apparent K(d) of 0.51-1.81 nM for factor Va). These results indicate that the prothrombinase complex forms specifically on various extravascular cells. Although the prothrombinase complex can be assembled on monocytes and lymphocytes, it is not known why these cells can activate prothrombin specifically. These cells which have the capacity for prothrombin activator activity could also activate factor X; i.e. cells with factor X activation activity were able to convert prothrombin. These observations suggest that thrombin was generated via two procoagulant activities; factor X activation and subsequent prothrombinase complex formation on the surface of these cells. This mechanism may explain the various pathological states involving or resulting from extravascular thrombin and fibrin formation.     

Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va

The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg306 and Arg506 in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg506 cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg306 and Arg506 and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 microm prothrombin, whereas half-maximal inhibition was obtained at 0.7 microm prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg306 sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.     

HD1, a thrombin-directed aptamer, binds exosite 1 on prothrombin with high affinity and inhibits its activation by prothrombinase

Incorporation of prothrombin into the prothrombinase complex is essential for rapid thrombin generation at sites of vascular injury. Prothrombin binds directly to anionic phospholipid membrane surfaces where it interacts with the enzyme, factor Xa, and its cofactor, factor Va. We demonstrate that HD1, a thrombin-directed aptamer, binds prothrombin and thrombin with similar affinities (K(d) values of 86 and 34 nm, respectively) and attenuates prothrombin activation by prothrombinase by over 90% without altering the activation pathway. HD1-mediated inhibition of prothrombin activation by prothrombinase is factor Va-dependent because (a) the inhibitory activity of HD1 is lost if factor Va is omitted from the prothrombinase complex and (b) prothrombin binding to immobilized HD1 is reduced by factor Va. These data suggest that HD1 competes with factor Va for prothrombin binding. Kinetic analyses reveal that HD1 produces a 2-fold reduction in the k(cat) for prothrombin activation by prothrombinase and a 6-fold increase in the K(m), highlighting the contribution of the factor Va-prothrombin interaction to prothrombin activation. As a high affinity, prothrombin exosite 1-directed ligand, HD1 inhibits prothrombin activation more efficiently than Hir(54-65)(SO(3)(-)). These findings suggest that exosite 1 on prothrombin exists as a proexosite only for ligands whose primary target is thrombin rather than prothrombin.